The role of cis-regulatory elements in the inheritance of transcriptional memory through mitosis.

顺式调节元件在通过有丝分裂遗传转录记忆中的作用。

• 批准号: 10751881
• 负责人: STEPHANIE NICOLE OPRESCU
• 金额: $ 6.87万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751881
• 关键词: Area; Behavior; Binding; Binding Proteins; Binding Sites; Biological Assay; CCAAT-Enhancer-Binding Proteins; Candidate Disease Gene; Cell division; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; DNA Sequence; DNase I hypersensitive sites sequencing; Data; Deposition; Development; EP300 gene; Elements; Endoderm; Enhancers; Ensure; Exclusion; GATA1 gene; GATA4 gene; Gene Expression; Genes; Genetic Transcription; Genome; Goals; HNF4A gene; Histone H2B; Human Cell Line; Inherited; Interphase; Knowledge; Liver; Maintenance; Measurement; Measures; Mediating; Memory; Methods; Mitosis; Mitotic; Mitotic Activity; Molecular; Mutagens; Physical condensation; Process; Proteins; Regulatory Element; Research; Residual state; Resolution; Role; Specific qualifier value; TATA-Box Binding Protein; Therapeutic; Trans-Activators; arm; cell fate specification; cell type; daughter cell; genome-wide; histone modification; human embryonic stem cell; improved; insight; preservation; programs; promoter; recruit; small molecule; stem cells; transcription factor; transmission process

PROJECT SUMMARY The goal of this proposal is to determine how cell identity, as defined by a transcriptional program, is inherited through mitosis by systematically evaluating the role of specific cis-regulatory elements and trans- acting factors. The central hypothesis is that cis-regulatory elements at gene promoters maintain transcriptional competency through mitosis while trans interactions with enhancers mediate the timely and appropriate level of gene expression during mitotic exit. This proposal will leverage how the underlying DNA sequence determines the multifaceted interactions at promoters and cell-type specific enhancers that ensure cells faithfully re- establish proper transcriptional programs. This will be evaluated in cells with a stable identity and expanded to understand how this is mediated in the context of early development, when cells undergo constant fate decisions. While it was previously thought that chromatin condensation during mitosis excluded most proteins and thereby transcription-related processes, recent studies originally emanating from the Zaret lab now indicate that transcription is active at a low level and promoters, but not most enhancers, are accessible and maintain active histone modifications [1-7]; and mitotic chromatin arms retain areas of dynamic chromatin [8]. Although genome-wide measurements of histone modifications, chromatin accessibility and chromatin organization suggest mitotic changes at various cis-regulatory elements, they are unable to garner high- resolution mechanistic insight into how mitotic memory is retained at promoters and acted upon, during mitotic exit, by enhancers. While components involved in transcription such as the TATA-binding protein TBP, the elongating form of RNAP2, and transcription factors (TFs) such as GATA1, FOXA1, and ESRRB are detected on mitotic chromatin; many of these TFs do not remain bound to their interphase enhancer targets, thus how these factors function at specific loci to transmit transcription memory through mitosis remains elusive [5-7, 9- 13]. Therefore, this proposal aims to determine how cell fate is preserved through cell division by first evaluating the functional requirement for promoters in mitotic transcription, and then the mechanisms employed by liver-specific transcription factors to mediate enhancer activation upon mitotic exit. This will be assessed in the liver-derived HUH7 human cell line, which is highly amenable to mitotic synchronization methods, and expanded to determine the role of identified factors in pluripotent and endoderm-differentiated human embryonic stem cells. This proposal will expand our understanding of the molecular mechanisms that maintain cell identity and cell fate specification, thereby improving our ability to target and modulate stem cells for therapeutic value.

项目摘要 这个提议的目标是确定细胞的身份，如转录程序所定义的，是如何在细胞中表达的。 通过系统评估特定顺式调节元件和反式调节元件的作用， 作用因素核心假设是基因启动子上的顺式调控元件维持转录水平， 能力通过有丝分裂，而反式相互作用与增强介导的及时和适当的水平， 有丝分裂退出时的基因表达。这项提案将利用潜在的DNA序列如何决定 启动子和细胞类型特异性增强子之间的多方面相互作用，确保细胞忠实地重新 建立适当的转录程序。这将在具有稳定身份的细胞中进行评估，并扩展到 了解这是如何介导的背景下，早期发展，当细胞经历恒定的命运 决策虽然以前认为有丝分裂期间染色质凝聚排除了大多数蛋白质 因此转录相关的过程，最近的研究最初来自Zaret实验室， 表明转录在低水平下是有活性，启动子，但不是大多数增强子，是可接近的， 保持活跃的组蛋白修饰[1-7];有丝分裂染色质臂保留动态染色质区域[8]。 虽然组蛋白修饰、染色质可及性和染色质可染性的全基因组测量 组织表明在各种顺式调节元件的有丝分裂变化，它们无法获得高水平的 有丝分裂记忆是如何在启动子处保留并在有丝分裂过程中起作用的 出口，通过增强剂。虽然参与转录的组分如TATA结合蛋白TBP， RNAP 2的延伸形式，以及转录因子（TF）如GATA 1、FOXA 1和ESRRB被检测 在有丝分裂染色质上;这些TF中的许多不保持与它们的间期增强子靶结合，因此如何 这些因子在特定的基因座上发挥作用，通过有丝分裂传递转录记忆，这仍然是不清楚的[5-7，9- 10]。 13]。因此，这项建议旨在确定细胞命运是如何通过细胞分裂保存的， 评估启动子在有丝分裂转录中的功能需求， 由肝脏特异性转录因子使用，以介导有丝分裂退出时的增强子激活。这将是 在肝脏来源的HUH 7人细胞系中进行了评估，该细胞系非常适合有丝分裂同步化 方法，并扩大，以确定所确定的因素在多能和内胚层分化 人类胚胎干细胞这一建议将扩大我们对分子机制的理解， 维持细胞特性和细胞命运特化，从而提高我们靶向和调节干细胞的能力 治疗价值。